Techniques for studying single cells have become indispensable in cell biology for their ability to identify characteristics and behaviors that would otherwise be hidden using population averaged measures. As single-cell techniques continue to develop, these techniques have the potential to significantly impact many different areas of study. For example, the study of virus infections and virus-host interactions are particularly well-suited for such techniques. Virus infections are generally rapid and dynamic events that exhibit high levels of heterogeneity stemming from multiple sources. Thus, at any given time during an infection, different cells can respond with phenotypically different behavior and progress at different times and rates, making it difficult to use average readouts to make inferences concerning the sequence or timing of infection events or for relating changes in one biological measure to another.
The most basic advantage of single cell data for addressing these challenges is the ability to categorize a heterogenous group of individual cells into cohorts or subpopulations with similar individual characteristics to explore the potential relationship of those characteristics to heterogenous outcomes. In other words, the heterogeneous system behavior can be leveraged to learn more about important cellular characteristics. The most prominent example of this is the use of flow cytometry, where multiple fluorescent tags or reporters can be simultaneously quantified for each cell in a population of thousands to provide exquisite, quantitative insight into the presence and nature of subpopulations. However, this powerful tool is often difficult to apply in the area of virology given the danger of contamination and production or aerosolized virus on shared flow cytometry equipment. Flow cytometry is also typically limited to endpoint analysis. Although many other single cell techniques have been developed such as droplet-based microfluidics and microfluidic flow traps, sandwiched microwells (SMAs) offer an attractive alternative with respect to flexibility, throughput, cost, and required expertise for operation. Further, SMAs offer the capability to observe single cell behavior over time.
As is known, a SMA is a sandwiched structure that is formed from a first plate with an array of microwells formed therein and a second plate that acts as a lid. When sandwiched together, the microwells and the lid create sealed chambers in which a screening reaction can be carried out. It can be appreciated that the use of microwells (wells on the order of ˜1-200 μm) is prevalent in microscale device design primarily to help isolate analytes into very small volumes. By doing this, assays can be made vastly more sensitive and can be massively parallelized. Although microwells can be used to isolate small volumes of liquid for screening, they are extremely useful for isolating individual or small numbers of particles or molecules suspended in that liquid or fluid for independent analysis. These types of advantages drive much of the current research in the area of microfluidics in general. The reduced volumes of the analytes allow for more sensitive detection of proteins and other molecules given that when these molecules are produced in a microwell (100×100×100 μm=1 nano liter) during a reaction or cell culture, they are diluted into much less volume than that of a more standard reaction or culture vessel, such as a 96 well plate (200 micro liters). Consequently, a 200,000 fold reduction in volume produces a 200,000 fold increase in the concentration of the produced molecule. The increase in the concentration of the produced molecule greatly increases the ability to detect such production.
Heretofore, however, methods to interface with and leverage microwells with these types of dimensions have been limited. Current embodiments of SMAs allow only a single experimental condition to be examined per chip, thereby making it difficult to control for chip-to-chip differences. Further, current methods for loading and treating the microwells, although generally easy, are relatively difficult to control and standardize.
Therefore, it is primary object and feature of the present invention to provide a microwell device for isolating a fluid, such as an analyte, into very small volumes.
It is a further object and feature of the present invention to provide a microwell device for isolating a fluid into very small volumes which is simple to utilize and inexpensive to manufacture.
It is a still further object and feature of the present invention to provide a microwell device for isolating a fluid into very small volumes which may be used in combination with conventional micropipetting equipment.
In accordance with the present invention, a microwell device is provided. The device includes a plate having a upper surface with a plurality of microwells formed therein. The microwells are adapted for receiving a fluid therein. A barrier extends about a first portion of the microwells. The barrier prevents fluid deposited on the first portion of the microwells from flowing therepast.
A recess formed in the upper surface of the plate within the barrier. The recess has an outer periphery and the first portion of microwells are spaced about the outer periphery of the recess. The recess has a volume and each of the microwells also has a volume. The volume of the recess is greater than the volumes of the microwells.
By way of example, the barrier may be a channel formed in the upper surface of the plate. The channel has a volume which is greater than the volumes of the microwells. The barrier is generally circular. The barrier may be a first barrier and the device may also includes a second barrier extending about a second portion of the microwells. The second barrier prevents fluid deposited on the second portion of the microwells from flowing therepast.
In accordance with a further aspect of the present invention, a microwell device is provided. The device includes a plate having a upper surface with a plurality of microwells formed therein. The microwells are adapted for receiving a fluid therein. First and second recesses may also be formed in the upper surface of the plate. Each recess has an outer periphery. A first portion of microwells are spaced about the outer periphery of the first recess and a second portion of microwells are spaced about the outer periphery of the second recess.
A first barrier may be positioned between the first and second portions of microwells for fluidicly isolating the first portion of the microwells from the second portion of microwells. In addition, a second barrier may also be positioned between the first and second portions of microwells for fluidicly isolating the second portion of the microwells from the first portion of microwells. The first barrier may take the form of a first channel in upper surface of the plate that extends about the first portion of microwells. The first channel may have a generally circular configuration. It is contemplated for the first channel to have a volume and for each of the first portion of microwells has a volume. The volume of the first channel is greater than the volumes of each of the first portion of microwells. The second barrier may take the form of a second channel in upper surface of the plate that extends about the second portion of microwells.
It is intended for the first and second recesses to have volumes and for each of the first and second portions of microwells to have a volume. The volume of the first recess is greater than the volumes of each of the first portion of microwells and the volume of the second recess is greater than the volumes of each of the second portion of microwells.
In accordance with a still further aspect of the present invention, a microwell device is provided. The device includes a plate having a upper surface. The upper surface includes first and second recesses formed in the upper surface of the plate. Each recess has an outer periphery. A first portion of microwells is formed therein in the upper surface of the plate. The first portion of microwells is spaced about the outer periphery of the first recess. A second portion of microwells is also formed in the upper surface of the plate. The second portion of microwells spaced about the outer periphery of the first recess. A first barrier extends about the first portion of the microwells for fluidicly isolating the first portion of the microwells and a second barrier extends about the second portions of microwells for fluidicly isolating the second portion of the microwells.
The first barrier includes a first channel extending about the first portion of microwells. The first channel has a generally circular configuration and a volume. Each of the first portion of microwells also has a volume. The volume of the first channel is greater than the volumes of each of the first portion of microwells. The second barrier includes a second channel extending about the second portion of microwells. The first and second recesses have volumes and each of the first and second portions of microwells have a volume. The volume of the first recess is greater than the volumes of each of the first portion of microwells and the volume of the second recess is greater than the volumes of each of the second portion of microwells. A lid having a surface may also be provided. The lid is moveable between a first position wherein the surface of the lid is spaced from the upper surface of the plate and a second position wherein the surface of the lid is in engagement with the upper surface of the plate.